Safire2 fluorometer

Measurement of GLP-1 was done in duplicate by following the procedure of GLP-1 ELISA assay kit of EMD-Millipore, USA. Anti-GLP-1 monoclonal antibodies were already coated in the 96-well microtiter plate. The standards, QC 1, QC 2 and samples were added after assay buffer in respective wells, mixed gently and the plate was incubated at 4 °C for 20–24 h. On next day detection conjugate was added, incubated for 2 h and after washing three times with wash buffer, the substrate was added in each well. After 20 min incubation stop solution was added to stop the reaction and incubated again for a further 5 min at room temperature in the dark to arrest phosphatase activity. The fluorescence was measured using Excitation and Emission wavelength at 355 and 460 nm respectively with the help of the Tecan Safire² fluorometer (Reading, England, UK). The amount of GLP-1 was determined in the sample from the standard curve constructed with standards provided along with the kit. For GLP-1 secretion studies, some selected fractions with good enzyme inhibitory action were evaluated at a single concentration of 1 mg/ml.

Secretase activity assays were performed, as described before [82 (link),83 (link)]. Following the incubation, cells were washed twice with prewarmed imaging solution containing 140 mM NaCl, 5 mM KCl, 8 mM CaCl₂, 1 mM MgCl₂, 20 mM HEPES, pH 7.4. Afterwards, 100 μL (α)/50 μL (β and γ) imaging solution mixed with 3 μM α-secretase substrate (Calbiochem, No. 565767), 20 μM β-secretase substrate (Calbiochem, No. 565758) or 6,25 μM γ-secretase substrate (Calbiochem, No. 565764) was added. The resulting fluorescence was determined continuously at excitation wavelengths of 340 ± 10 nm (α), 345 ± 5 nm (β), 355 ± 10 nm (γ) and emission wavelengths of 490 ± 10 nm (α), 500 ± 5 nm (β), 440 ± 10 nm (γ) under light preclusion and at 37 °C in a Safire2 Fluorometer (Tecan, Crailsheim, Germany).

Measurement of NEP activity was performed as published in Miners et al. [84 (link)], with minor modifications utilizing the anti-NEP antibody AF1182 (R&D Systems, Minneapolis, MN, USA) and 5 μM MCA-RPPGFSAFK(DNP)-OH fluorogenic peptide substrate (R&D Systems, Minneapolis, MN, USA). Cells were chemical lysed in lysis buffer containing 0.5% TritonX-100, 20 nM Tris pH 7.4, 10% sucrose and fluorescence at an excitation wavelength of 320 ± 10 nm, and an emission wavelength of 405 ± 10 nm was measured in a Safire2 Fluorometer (Tecan, Crailsheim, Germany).

Ten μL of sample was added to 190 μL of ThT dissolved in 10 mM phosphate buffer, pH 7.4, and then the mixture was vortexed briefly. Fluorescence was determined three times at intervals of 10 s using a Tecan Safire 2 fluorometer. Excitation and emission wavelengths were 450 nm and 482 nm, respectively. Sample fluorescence was determined by averaging the three readings and subtracting the fluorescence of a ThT blank.

IDE enzyme activity was analyzed as described in^{70 (link)} with minor modifications. Anti-IDE antibody ST1120 (Merck) was diluted in 1xPBS (5 µg/ml) and coated on a Nunc MaxiSorp 96-well plate for 24 hours at room temperature. After washing the plate five times with 1x PBS containing 0.5% Tween-20, blocking was performed by incubating 10% fatty acid free bovine serum albumin in 1x PBS for two hours at room temperature. Afterwards, plate was washed three times and lysates were incubated for one hour at 20 °C and 150 rpm, followed by five wash steps. After a pre-incubation with assay buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 10 µM MgCl₂, β-secretase inhibitor II (565749, Merck), γ-secretase inhibitor IV (565761, Merck), complete protease-inhibitor without EDTA) for 15 minutes at room temperature, the fluorogenic peptide substrate Mca-RPPGFSAFK(Dnp)-OH (10 µM) was added and fluorescent was measured using a Safire² Fluorometer (Tecan, Crailsheim, Germany) at an excitation wavelength of 320 ± 10 nm, and an emission wavelength of 405 ± 10 nm.

Cells were seeded on black 96-well plates (2 × 10⁴ Neuro2a cells/ well, 3 × 10⁴ SH-SY5Y cells/ well) and treated as described above.
For determination of H₂O₂ accumulating in the medium during the treatment period, 50 µl conditioned cell culture supernatant of each well were transferred to a new 96-well plate and supplemented with 5 µM Amplex Red (Cayman Chemical, Ann Arbor, USA) and 0.01 U/ml HRP (Thermo Fischer Scientific, Schwerte, Germany). A potential interference between the added agents and the HRP-catalyzed reaction between Amplex Red and H₂O₂ was analyzed by repeating this experiment in a cell-free system utilizing freshly prepared DMEM/0.1% FCS + phospholipids (10 µM) or solvent in the presence of supplemented H₂O₂ (0.000007%).
For measuring the H₂O₂ freshly released by pretreated cells, the supernatant was removed and 50 µl of Amplex Red reaction mixture (5 μM Amplex Red and 0.01 U/ml HRP in phenol red-free DMEM/0.1% FCS) was added to each well.
The fluorescence signal of resorufin was determined at an excitation wavelength of 530 ± 17 nm and an emission wavelength of 590 ± 17 nm for 16 min (120 s (seconds) intervals) at 37 °C under light exclusion in a Safire² Fluorometer (Tecan, Crailsheim, Germany). The increase of fluorescence over time was calculated for each well and used for further data analysis.

Evaluation of β- and γ-secretase activity has been described in detail earlier [20 (link)]. Shortly, fluorogenic β-secretase substrate IV (Calbiochem, Darmstadt, Germany) or γ-secretase substrate (Calbiochem) was added in a final concentration of 20 or 10 µM to samples, respectively. In the case of β-secretase, the resulting fluorescence was measured continuously under light exclusion with a Safire2 Fluorometer (Tecan) at an excitation wavelength of 345 ± 5 nm and an emission wavelength of 500 ± 2.5 nm, for γ-secretase at an excitation wavelength of 355 ± 10 nm and an emission wavelength of 440 ± 10 nm.